Cervista HPV 16/18 REF 95.439

THIRD WAVE TECHNOLOGIES

An in vitro diagnostic test for the detection of DNA from Human Papillomavirus (HPV) Type 16 and Type 18 in Cervical Specimens.

IVDi96I . 3.,c

In vitro diagnostic Contains sufficient Temperature Limitation medical device reagents for 96 tests

Do NOT store in frost-free freezer. Protect from light.

1 of 39 TABLE OF CONTENTS NAME AND INTENDED USE ABBREVIATIONS USED SUMMARY AND EXPLANATION OF THE TEST PRINCIPLES OF THE PROCEDURE REAGENTS PROVIDED WARNINGS AND PRECAUTIONS REAGENT STORAGE AND HANDLING REQUIREMENTS ADDITIONAL REAGENTS AND MATERIALS MATERIALS REQUIRED, BUT NOT PROVIDED SPECIMEN COLLECTION, DNA EXTRACTION AND STORAGE FOR ANALYSIS TEST PROCEDURE PROCEDURAL NOTES AND PRECAUTIONS INTERPRETATION OF RESULTS QUALITY CONTROL LIMITATIONS PERFORMANCE CHARACTERISTICS REFERENCES TROUBLESHOOTING GUIDE

2 of 39 - ' NAME AND INTENDED USE

The Cervista Tm HPV 16/18 test is an in vitro diagnostic test for the qualitative detection of DNA from Human Papillomavirus (HPV) Type 16 and Type 18 in cervical specimens. The Cervista Tm HPV 16/18 test uses the Invader® chemistry, a signal amplification method for detection of specific nucleic acid sequences. This method uses two types of isothermal reactions; a primary reaction that occurs on the targeted DNA sequence and a secondary reaction that produces a fluorescent signal (See Figure 1).

The Cervista Tm HPV 16/18 test is indicated:

1) In women 30 years and older the Cervista TM HPV 16/18 test can be used adjunctively with the CervistarMI HPV HR test in combination with cervical cytology to assess the presence or absence of high-risk HPV types 16 and 18. This information, together with the physician's assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management.

2) To be used adjunctively with the Cervista TM HPV HR test in patients with atypical squamous cells of undetermined significance (ASC-US) cervical cytology results, to assess the presence or absence of high-risk HPV types 16 and 18. This information, together with the physician's assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management. The results of this test are not intended to prevent women from proceeding to colposcopy.

Cervical specimens that may be tested with the Cervista T HPV 16/18 test include the following preservation system and collection devices:

• ThinPrep® Pap Test PreservCyt® Solution * Broom-type device (e.g. Rovers Cervex® Brush, Wallach Papette®), or Endocervical Brush/Spatula

WARNINGS * This test is not intended for use in determining the need for treatment (l~eexcisional or ablative treatment of the cervix) in the absence of high-grade cervical dysplasia. Patients who are HPV 16/18 positive should be monitored carefully for the development of high-grade cervical dysplasia according to current practice guidelines. * The Cervista Tm HPV 16/18 test is not intended for use as a stand-alone assay. Results should be interpreted in conjunction with the CervistaTm HPV HR and cervical cytology test results. * The Cervista Tm HPV 16/18 test is not intended for use in women under age 30 with normal cervical cytology. * The Cervista Tm HPV 16/18 test is not intended to substitute for regular cervical cytology screening. * The use of this test has not been evaluated for the management of women with prior cytological or histological abnormalities, hysterectomy, who are pregnant, 3 of 39 postmenopausal, or who have other risk factors (e.g. HIV+, immunocompromised, history of STI).

The CervistaTM HPV 16/18 test is designed to enhance existing methods for the detection of cervical disease and should be used in conjunction with clinical information derived from other diagnostic and screening tests, physical examinations, and full medical history in accordance with appropriate patient management procedures.

CervistaTM HPV 16/18 test results should not be used as the sole basis for clinical assessment and treatment of patients.

ABBREVIATIONS USED

ASC-US: Atypical squamous cells of undetermined significance LSIL: Low-grade squamous intraepithelial lesion CIN: Cervical intraepithelial neoplasia CLSI Clinical and Laboratory Standards Institute DNA: Deoxyribonucleic acid FAM: Carboxyfluorescein dye Red: Redmond® red dye FRET: Fluorescence resonance energy transfer FOZ: Fold over zero (sample or control signal divided by No Target Control signal) gDNA: Genomic DNA HIST2H2BE: Human histone 2 gene, H2be gene HPV: Human papillomavirus HR: High-risk LoB Limit of Blank LoD Limit of Detection Max. Maximum Min. Minimum NILM Negative for intraepithelial lesion or malignancy. This category encompasses the previous categories of "within normal limits" and "benign cellular changes". NTC: No target control Oligo: Oligonucleotide Pap: Papanicolaou cervical cytology test RFU: Relative fluorescence unit TWT: Third Wave Technologies, Inc.

SUMMARY AND EXPLANATION OF THE TEST Over 100 HPV types have been documented in the literature, approximately 40 of which infect the anogenital area and are transmitted sexually. Anogenital HPV is associated with virtually all cancers of the cervix.' Cervical cancer has previously been shown to be highly preventable when cytological and HPV screening programs are employed to facilitate the detection and treatment of pre-cancerous lesions.

4 of 39 Of the sexually transmitted types of HPV, 14 oncogenic genotypes (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), are considered high-risk (HR) HPV types due to their strong association with cervical cancers (relative to low risk HPV types, which have little or no association with cervical cancer). 2 ,'19 Still, the vast majority of high-risk HPV infections are cleared 4 Very few high-risk HPV DNA positive women develop cytologic high-grade SIL (HSIL) indicating underlying CIN 2-3 or cancer. 5 The absolute risk of developing an incident cytologic abnormality following a HR HPV infection is known to vary in different populations. The presence of high-risk HPV DNA in conjunction with an equivocal or ambiguous cytology result (ASC-US) places a woman at increased risk for having an underlying cervical intraepithelial neoplasia 2 or 3 (CIN2 or CIN3).6 '7' 8 CIN3, while occurring in only approximately 5% of ASC-US cases, 9 is an immediate precursor to cervical cancer and consequently its detection is very important for patient management. 3,7 Therefore, the identification of those women with ASC-US cytology in conjunction with a high-risk HPV infection is a useful aid for clinicians to decide who should be monitored or treated more aggressively. 3' 810 1,' 1,12,13 Current scientific literature suggests that persistent infection with high-risk HPV is the main risk factor for development of high-grade cervical neoplasia and cancer.4 5' '15 Apparent persistence may represent continuous infection with a single HPV type, with multiple HPV types, or reinfection. Nonetheless, women with normal cervical cytology who are HR HPV negative appear to be at low risk for having or developing cervical precancerous lesions.' 10 HPV types 16 and 18 are recognized as both highly oncogenic and persistent, associated with 60% and 10% of cervical cancers, respectively, while also having the lowest clearance rates in cervical screening. 10' 1 4' 1 5'16 As a result, numerous studies report that women infected with HPV types 16 and 18 have a significantly higher risk for developing ->CIN3 than women infected with other high-risk types . 31 0,11 ,13 Beginning in 2002, patient management guidelines have been published by various groups of U.S. healthcare professionals that recommend how women should be screened for cervical cancer according to age, the presence of cytological abnormalities in a cervical sample, and other factors. 71' ' 19 These patient management guidelines consistently recommend testing for the presence of high-risk types of HPV as a regular screening tool, in combination with cytology in two instances: 1) for women age 30 and over; and 2) as an additional diagnostic tool for women 21 years of age and over with ASC-US.

According to the 2006 Consensus Guidelines for the Management of Women with Abnormal Cervical Cancer Screening Tests, women with negative cervical cytology results who test positive for the presence of high-risk HPV DNA would benefit from type-specific HPV genotyping. 1" 9 Studies support the validity of genotyping for HPV types 16 and 18, whether it is used as a follow-up to a high-risk HPV screening test or performed concurrently. 12 3', 10 , 31,9 ,20

PRINCIPLES OF THE PROCEDURE Cervista HPV 16/18 is a qualitative, in vitro diagnostic test for the detection of DNA from two high-risk HPV types: 16 and 18. The CervistaTM HPV 16/18 test uses the Invader® chemistry, a signal amplification method for detection of specific nucleic acid sequences. The Invader® technology uses two types of isothermal reactions: a primary reaction that occurs on the targeted DNA sequence and a secondary reaction that produces a fluorescent signal (See Figure 1). In the primary reaction, two types of sequence specific oligonucleotides (i.e. a probe oligonucleotide and an Invader® oligonucleotide) bind to the DNA target sequence. When these oligonucleotides overlap by at least one base pair on the target sequence, an invasive

5of39 Li/' structure forms that acts as a substrate for the Cleavase® enzyme. The enzyme cleaves the 5' portion (flap) of the probe at the position of the overlap. The probes are present in large molar excess and cycie rapidly on and off the target sequence so that many cleaved 5' flaps are generated per target sequence. The cleaved flaps then bind to a universal hairpin FRET oligonucleotide creating another invasive structure that the Cleavase® enzyme recognizes as a substrate. The enzyme cleaves the FRET oligonucleotides between the fluorophore and quencher molecule and produces fluorescence signal as the cleaved flaps cycle on and off. For each copy of target, the combined primary and secondary reactions result in 106~_iO0 fold signal amplification per hour .21 The flap sequences and FRET oligonucleotides are universal since they are not complementary to the targeted sequence. The reagents for this test are provided as two oligonucleotide mixtures, which detect HPV16 and HPV1S. Oligonucleotides that bind to the human histone 2 gene (H2be, HlST2H2BE) are also present in these two oligonucleotide mixtures. HlST2H2BE serves as an internal control producing a signal from cellular DNA present in the sample. The format of the Cervista TM HPV 16/18 test allows simultaneous detection of HPV DNA sequences and HIST2H2BE in a single well by utilizing two different 5'-flap sequences on the probes as well as two different FRET oligonucleotides, each with a spectrally distinct fluorophore (FAM and Red). By design, the released 5'-flaps bind only to their respective FRET oligonucleotides to generate target-specific signal (see Figure 1). A positive result for HPV16, HPV18 or HPV16 and HPV18 is represented by a FAM fluorescent signal that lies above an empirically derived cut-off value. For each reaction, a negative result is represented by a FAM fluorescent signal that lies below the same empirically derived cut-off value. As a means to determine the relative quantity of sample DNA in each reaction, Human HlST2H2BE is measured by a Red fluorescent signal that lies above an empirically derived cut-off value in each reaction. The measure of this target serves as a quality control mechanism to confirm that a negative result is not due to insufficient sample. This internal control target also serves as an internal processing measure to ensure that the testing procedure has been adequately performed.

6 of 39 7 la. HPVoligos form invasive lb. HIST2H2BEoiigos form invasive structure on HPV DNA. structure on qenorric DNA.

2. Cleavastf enzyme recognizes structure and cleaves probe oligos

3a. Flaps from HPV probe oligos form 3b. Flaps from HIST2H-213E probe oligos form invasive structure on FAM FREToligos invasive structure on Red FRET oligos

__ __

4. Cleavasd? enzyme recognizes structure and releases fluorophores from FRET oligos creating fluorescence signal.

FAM Red Fluorescence 44 Fluorescence

Figure 1: A graphic representation of the Invader® chemistry in the Cervista TM HPV 16118 test.

7 of 39 REAGENTS PROVIDED Table 1: CervistaTM HPV 16/18 REF 95-439) Contents Vial Label Vial Quantity Reagent Abbreviation & Reagent Description Volume HPV16 016 Oligonucleotides with affinity to HPV type (Red cap and red 1 x 1400 pL 16 and human HIST2H2BE suspended in stripe) water and MOPS buffer (pH 7.5) HPV18 018 Oligonucleotides with affinity to HPV type (Teal cap and teal 1 x 1400 pL 18 and human HIST2H2BE suspended in stripe) water and MOPS buffer (pH 7.5) Cleavase® E Cleavase® Enzyme suspended in 140 mM MgCI2, 10 mM Tris (pH 8.0), 25 mM KCI, Enzymeon (Prl cpad 10 L 0.25% Tween 20, 0.25% Nonidet P40, 25% Solutionpurple stripe) Glycerol and 0.05 mg/mL BSA C16 500 copies/pL cloned HPV type 16 DNA HPV16 and 3000 copies/pL cloned HIST2H2BE Control (Clear cap and 1 x 200 jiL DNA in yeast tRNA and 10 mM Tris, 0.1 mM black stripe) EDTA Buffer C18 500 copies/pL cloned HPV type 18 DNA HPV18 and 3000 copies/pL cloned HIST2H2BE Control (Clear cap and I x 200 pL DNA in yeast tRNA and 10 mM Tris, 0.1 mM black stripe) EDTA Buffer NTC No Target Yeast tRNA and 10 mM Tris, 0.1 mM EDTA Control (Clear cap and 1 x 200 pL Buffer black stripe)

WARNINGS AND PRECAUTIONS For in vitro diagnostic use.

Safety and Handling Precautions 1. Universal safety precautions should be used when handling any human tissues or fluids. Specimens should be disposed according to local requirements. 2. Product components (product residuals, packaging) can be considered as laboratory waste. Dispose of unused reagents and waste in accordance with applicable federal, state, and local regulations.

REAGENT STORAGE AND HANDLING REQUIREMENTS * Store all reagents between -30°C and -15°C. * Do not use reagents past expiration date indicated on outside of package. * Do not store in a "frost-free" freezer. * Protect from light.

8 of 39 L/ * Prior to use, remove reagents from freezer and allow them to thaw at least 30 minutes at room temperature or until visual inspection indicates that no frozen material is present. * Vortex reagents prior to each use. * Third Wave Technologies recommends no more than ten (110) freeze-thaw cycles for all Cervista TmHPV 16/18 test reagents.

ADDITIONAL REAGENTS AND MATERIALS Invader Call Reporterm software is a required component of this IVOD test. This software is provided once with the initial order of the CervistaTM HPV 16/18 test and, thereafter, when incremental updates to the software are released. The Genfind Tm DNA Extraction Kit is an accessory of the CervistaTm HPV 16/18 test. Contact Third Wave Technologies to order the Genfind Tm DNA Extraction Kit (REF 95-449).

MATERIALS REQUIRED, BUT NOT PROVIDED

Consumable Supplies * Pipette tips, filter barrier and nuclease-free * 96-well polypropylene plates * Clear piate sealers * Mineral oil, molecular bioiogy grade * 2.0 mL sterile polypropylene tubes and screw caps

Equipment * Pipettes * Vortex * Fluorescence plate reader (See Table 3) * Desktop PC with Microsoft® Windows® XP operating system with Microsoft® Excel' and Adobe® Reader® software * Thermal cycler or oven capable of maintaining recommended reaction temperatures

SPECIMEN COLLECTION, DNA EXTRACTION, AND STORAGE FOR ANALYSIS Cervical specimens should be collected in PreservCyt® Solution, the ThinPrep® Pap Test preservation system, using a broom-type device (e.g. Rovers Cervexe Brush, Wallach Papette®), or Endocervical Brush/Spatula.

9Sof 39 For Cervista TMHPV 16/18 testing, cervical specimens can be stored at room temperature (20-300C) in PreservCyt® Solution for up to 18 weeks prior to performing the test. Preservoyt® Solution specimens cannot be frozen. DNA should be extracted from Preservoyt® specimens using the Genfind TM DNA Extraction Kit (REF] 95-449).

TEST PROCEDURE Note: Perform DNA extraction from cervical specimens collected in Preservoyt® solution using the Genfind TM DNA Extraction Kit (FREF7 9549 prior to beginning the reaction procedure. Residual DNA extracted as part of the CervistalmH PV HIR test may be used for Cervista TM HPV 16/18 testing.

Reaction Procedure 1. Add 10 pL of each control and sample DNA to two wells of a 96-well plate as indicated in the test plate layout (see Figure 2).

PV6 P1 HV6 PV8HPI P1HPVPVI6 HPM,i8V HPVIB PH18' H-PVI6 lV" Mix Mkf Mix

Table 2: Reaction Mix Preparation Instructions

Reagent piLlReaction No. of Reactions TtlVlm ______(Samples & Controls (k)) T tl V l m HPV Oligo Mix 16 or 18 8pjL k =8k(l.25)p/L

Gleavase® Enzyme Solution 2pjL k =2k(l.25) pL

Total Mix Voiume 10 p1L k =l0k(1.25) pL

10 of 39 6. Decrease thermal cycler temperature setting to 63CC. 7. Add 10 piL of the appropriate reaction mix to each well containing a control or sample (see Figure 2), taking care to pipette below the mineral oil. 8. Incubate the plate at a 6300 setting for 4 hours. Data Collection 1. Always bring the plate to room temperature before reading. If the plate cannot be read immediately, store it at 2-8oC (it is recommended to read the plate within 24 hours of test completion). 2. Place the 96-well plate (well Al must be in upper left corner) in the plate holder of the fluorescence plate reader. Remove plate-sealing tape. 3. Define the plate type to set up the coordinates and probe height for the specific type of plate. Save the settings. 4. Read the entire plate. Two separate scans are required; FAM (Excitation = 485 nm, Emission = 535 nm) and Red (Excitation = 560 nm, Emission = 612 nm). To detect the HPV signal, the instrument should be set to detect the FAM dye first. To detect the sample genomic DNA, the instrument should be set to detect the Red dye. 5. Adjust the gain of the fluorescence plate reader to be in the linear dynamic range of the reader according to the manufacturer's instructions. The gain should be set so that the No Target Control (NTC) yields values that are in the background range of the reader, with a minimum RFUI of 600. The NTC values do not have to be identical for the FAM and Red reads. Table 3: Fluorescence Plate Reader Specifications/Settings Multi-Labeling Measurement Measurement I Measurement 2 Parameters(AMRe Read Made: Top Top Excitati~on~4820n5620m wavelength/Bandwidth:48/0n5020m Emission wavelength/Bandwidth:535/25 nmn 612/1 0 nm Number of flashes: 010 1

Integration time: 2Ops 20ps

PROCEDURAL NOTES AND PRECAUTIONS 1.Laboratories should use good laboratory practices and comply with all applicable federal, state and local regulatory requirements. 2. Do not pool reagents from different lots or from different vials of the same lot. These components have been tested as a unit. Do not interchange components from other sources or from different lots. 3. Do not use reagents after their expiration date. 4. Mix the samples, reagents, and reaction mixes thoroughly and consistently. 11 of 39 8- 5. Use nuclease-free, sterile disposable aerosol barrier pipette tips for each addition and transfer to avoid cross-contamination. 6. Use nuclease-free, disposable polypropylene tubes for preparing the reaction mixes. 7. Verify that the 96-well plate type is compatible with the specific thermal cycler and fluorescence plate reader to be used before starting the test. 8. Controls must be added to the designated positions on the test plate layout shown in Figure 2 in order for the Invader Call Reporter TM software to function properly. 9. Use fresh mineral oil for each reaction setup (do not transfer these reagents back to the original container once they have been dispensed). 10. Refer to the test plate layout to ensure that the correct mix is added to the appropriate column. 11. Always place the pipette tip near the bottom of the well to ensure that the reaction mix is added below the mineral oil. Mix by carefully filling and emptying the pipette tip 3-5 times. 12. The Cervista TM HPV 16/18 Test Procedure, Quality Controls, and the Interpretation of Results must be followed closely to obtain reliable test results.

INTERPRETATION OF RESULTS

A signal to noise value (sample signal measured against signal from a No Target Control reaction well) is referred to as FOZ (Fold-Over-Zero). FOZ values are generated for both the HPV 16 and HPV 18 reactions. A final positive, negative or indeterminate result for any particular sample is generated based on the analysis of two separate reaction wells. When the HPV1 6 FOZ value and/or HPV1 8 FOZ value is greater than 2.13, the sample is positive for HPV 16 and/or HPV 18.

An indeterminate call is generated in three different scenarios 1) when the % difference between the gONA FOZ values is Ž:25.0% (High % difference), 2) when both HPV FOZ values are < 0.7 (Low HPV FOZ) and 3) when average gDNA FOZ of a negative sample is < 1.5 (low gDNA). An indeterminate call is indicative of insufficient mixing, a pipetting error or inadequate gDNA in the sample (see Troubleshooting Guide). A summary of the sample call criteria described above is shown in Figure 3. Terminology HPV FOZ: For each HPV Oligo Mix, the FAM signal of the sample divided by the FAM signal of the No Target Control. Average gDNA FOZ: The average value determined from the two genomic DNA FOZ values obtained from both of the reaction mixes, calculated by dividing the Red signal of the sample by the Red signal of the No Target Control. %Difference aDNA FOZ: The absolute value of the difference between the HPV16 and HPV18 genomic DNA FOZ values divided by the average genomic DNA FOZ value of the two HPV Oligo Mixes.

12 of39 1)3 Criteria Results Generated

%Difference genomic NO DNA < 25% "IND:High %Difference"

I YES

One or more HPV FOZ NO "IND: Low HPV FOZ" values > 0.70

YES

HPV16 FOZ <2.13 NO HPV18FOZ<2.13 . "POS:HPVI6&18"

YES

Average gDNA FOZ NO J"G < 1.50

YES

YES ______"iND: Low gDNA"

Figure 3: CervistaTM HPV 16118 Sample Call Criteria Ordered Top to Bottom

Note: The CervistaTM HPV 16/18 test does not require the use of an equivocal or re-test zone.

Table 4: Interpretation of Cervista TM HPV 16/18 Test Results when High-risk (HR) HPV Results are Positiveb. CervistaTM Interpretation for patients Interpretation for HPV16118 Test Result Report with NILM cytology who are patients with Result c ->30 years old8 ASC-US cytology POS:HPV 16 _KHPV type 16 Low but increased likelihood that detected underlying high-grade CIN will Increased likelihood that HPV type 18 be detected at colposcopy. underlying high-grade detected Medical literature suggests that CIN will be detected at POS: HPV16 & HPV types 16 and progression to high-grade colposcopy. HPV18 18 detected disease is possible. 3,10,11,19 Likelihood of underlying CIN2-3 or cancer is Low likelihood of underlying oter non-i6/ high ~ NE~dHPV types 16 CIN2-3 or cancer; results are not risk HPV t sill NEG dand/or 18 not intended to prevent women from confersVrisk.sRstill detected further cytology or HPV retes~ngYY0A¶19 are not intended to prevent women from proceeding to colposcop¥.

13of39 IND: High % CV Indeterminate HPV16/18 status unknown IND: Low gDNA aAccording to the 2006 consensus guidelines, women 30 years and older with greater than ASC-US cytology (including ASC-H, LSIL or above) should In cases where proceed to colposcopy regardless of their HPV test results. HPV HR and HPV 16/18 are run at the same time and a HR negative result is obtained alongside a 16/18 positive result, the 16/18 result is not interpretable. If both test results are negative, interpret the results the same as you would a HR negative TM result. 'The Cervista HPV 16/18 test does not determine whether high-risk HPV types other than 16/18 are present. An individual may be simultaneously infected with multiple HPV types. dIndividuals who are CervistaTM HPV HR positive and Cervista HPV 16/18 negative are most likely infected with a non-16/18 high-risk HPV type.

QUALITY CONTROL Internal Control The CervistaTM HPV 16/18 test includes an internal control which determines the relative quantity of sample DNA in each reaction. The internal control, Human HIST2H2BE, is measured by a Red fluorescent signal that lies above an empirically derived cut-off value in each reaction. The measure of this target serves as a quality control mechanism to confirm that a negative result is not due to insufficient sample. This internal control target also serves as an internal processing measure to ensure that the testing procedure has been adequately performed.

External Controls Negative Control The No Target Control must be run on each assay plate and results must meet the following criteria in order for the samples on that plate to be valid. If it does not meet these criteria, the samples and controls on that plate are invalid and must be repeated (see Table 5 for summary): 1. The minimum signal for each mix must be greater than or equal to 600 RFU (> 600). 2. The % Difference between the gDNA signals from both mixes must be less than 30.0% (<30.0%). Table 5: No Target Control Criteria

Result Min HPV Signal Min. gONA Signal Ma.%Dfenc (gDNA) Valid 600 600 29.9%

Positive Controls HPV controls (HPV16, HPV18) must be run on each assay plate and results must meet the following criteria for the test to be valid. If controls do not meet these criteria, the samples on that plate are also invalid and testing must be repeated (see Table 6 for summary). 1. The HPV FOZ value is determined by dividing the FAM signal of the control by the FAM signal of the No Target Control for each respective mix. The HPV16 Control should yield a positive HPV FOZ value (> 2.13) for only the HPV16 Oligo Mix and the HPV18 Control should yield a positive HPV FOZ value (->2.13) for only the HPV18 Oligo Mix.

14 of 39 -5 2. The average gDNA FOZ of the two mixes must be greater than or equal to 1.50 (Ž 1.50), or the control is invalid for low genomic DNA. 3. The % Difference between the gDNA FOZ values from both mixes should be less than 25.0% (<25.0%).

Table 6: HPV Control Criteria

ControlResult HPVI6 HPVI8 Average % Difference ControlResult FOZ FOZ gDNA FOZ gDNA FOZ valid >_21 21 .5<250 HPV16 Control Control Ž21 2.3 150<.%

HPV18 Control Cotrl S52.13 Ž2.13 Ž1.50 <25.0%

Note: Additional external controis may be tested according to guidelines or requirements of local, state, and/or country regulations or accrediting organizations. Any additional external controls should be tested in well(s) designated for patient samples per the plate layout.

Test Verification 1.Sample results are valid when the positive and negative controls yield the correct results. If the No Target Control (negative control) is invalid and/or any result for positive control(s) is invalid, all sample results on that plate are invalid and must be repeated. Refer to the Troubleshooting sections located in this insert and in the Software User Manual for Invader Call ReporterT m software. 2. All quality control requirements should be performed in conformance with local, state, and federal regulations as well as accreditations requirements.

LIMITATIONS 1. The Cervista TM HPV 16/18 test only detects DNA of HPV types 16 and 18. This test does not detect high-risk HPV types 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Nor does it detect HPV low-risk types since there is no known clinical utility for testing Low-Risk HPV types.19 2. The Cervista Tm HPV 16/18 test exhibits cross-reactivity to high levels of HPV High-Risk type 31. An HPV16 positive result was observed with 10o7 copies/reaction of HPV type 31. 3. A negative result does not exclude the possibility of HPV16 and/or 18 infection because very low levels of infection or sampling error may cause a false-negative result. 4. The test has been validated for use with cervical cytology specimens collected in PreservCyt® Solution using a Rovers Cervex Brush", Wallach Papette®, or Endlocervical Brush/Spatula. 5. The performance of the Cervista Tm HPV 16/18 test was established using DNA extracted with the Genfind Tm DNA Extraction Kit.

15 of39 6. The performance of the Cervista TM HPV 16/18 test was established using cervical cytology PreservCyt® specimens processed on the ThinPrep 2000 processor, it has not been established using other processors. 7. The performance of the CervistaTM HPV 16/18 test has not been adequately established for HPV vaccinated individuals. 8. Interference was observed in cervical specimens contaminated with high levels (2%) of contraceptive jelly and/or anti-fungal creams when DNA was isolated with the GenfindTM DNA Extraction Kit. Under these conditions, false-negative results may be obtained. 9. The Cervista TM HPV 16/18 DNA Test for human papillomavirus types 16 and 18 is not recommended for evaluation of suspected sexual abuse. 10. Prevalence of HPV infection in a population may affect performance. Positive predictive values decrease when testing populations with low prevalence or individuals with no risk of infection. 11. Infection with HPV is not an indicator of cytologic HSIL or underlying high-grade CIN, nor does it imply that CIN2-3 or cancer will develop. Most women infected with one or more high-risk HPV types do not develop CIN2-3 or cancer. 12.A negative HPV 16/18 result does not exclude the possibility of future cytologic HSIL or underlying CIN 2-3 or cancer. 13. PreservCyt® Solution specimens containing volumes less than 2 ml after the ThinPrep® Pap Test slides are prepared are considered inadequate for the CervistaTM HPV 16/18 Test.

EXPECTED RESULTS

High-Risk HPV16 and HPV18 Prevalence

The reported prevalence of HPV infection in women ranges widely, from 14% to more than 90%. 22 Several factors can affect the HPV prevalence among patient populations due to heterogeneity in geographic location, age, number of sexual partners, history of abnormal cervical cytology, coupled with differences in sampling techniques and testing methods and the intermittent nature of the infection. The CervistaTM HPV 16/18 multi-center prospective clinical study enrolled women from 89 clinical sites across 23 states throughout the United States which produced a demographically diverse patient population. Tables 7 and 8 show the prevalence of HPV 16 and HPV 18 observed in the study stratified by age.

Table 7: Prevalence of HPV16 and HPV18 Among Women with ASC-US Cytology Stratified by Age Age Group Prevalence of HPV 16 Prevalence of HPV 18 Prevalence of HPV 16&18 Positive 18 <21 31% (40/129) 7.8% (10/129) 1.6% (2/129) 21 < 30 23.7% (117/493) 6.3% (31/493) 1.6% (8/493) 30 < 39 11.9% (37/312) 2.6% (8/312) 0.6% (2/312) 39 < 49 8.3% (22/266) 1.9% (5/266) 0% (0/266) 49 < 59 5.9%(7/ 118) 1.7% (2/118) 0% (0/118) > 59 13.3% (6/45) 2.2%(1/45) 0% (0/45) All 16.8% (229/1363) 4.2% (57/1363) 0.9% (12/1363)

16 of 39 97 Table 8: Prevalence of HPV 16 and HPV 18 Among Women with NILMV Cytology Stratified by

A ge ______Age Group Prevalence of HPV 16 Prevalence of HPV 18 Prevalence of HPV 16118 30 <40 3.4% (21/616) 0.8% (5/616) 0.2% (1/616) 40 < 50 4.0% (27/674) 0.% 5/674)0(/64 50 <60 5.1% (25/486 1.0 (5/486)021/8 60 < 70 3.9% (6/154) 0%(0/154) 0%/ (0/1 54) >70 -0% (0/30) 0%(0/30) 0%(030 All 4.0% (79/1 960) 0.8% (15/1960 0.1% (2/1960

PERFORMANCE CHARACTERISTICS Clinical Sensitivity and Specificity of Cervista Tm HPV 16118 Among Women with ASC- US Cervical Cytology Results A multi-center prospective clinical study was conducted to evaluate the performance of the Cervista Tm HPV 16/18 test among patients with ASO-US cytology results to determine the need for referral to colposcopy. All clinical performance characteristics were established using ThinPrep liquid cytology specimens. Initial Thin Prep cervical specimens were classified according to the 2001 Bethesda System Classification. All women (18 years or older) with cytology results of ASC-US during routine cervical cancer screening procedures were invited to participate in the study prior to learning their HPV status. For women who consented, their initial residual ASO-US ThinPrep specimens were subsequently obtained for CerviStaTm HPV 16/18 testing. All patients who consented to the study underwent colposcopic examination. Investigators and patients remained blinded to the patient's HPV status until after completion of the colposcopic procedures, to avoid bias. Colposcopically directed histological specimens were examined by pathologists who were also blinded to the patient's HPV status. 1,514 women age 18 and over with ASC-US results were ultimately enrolled in the study from 89 clinical sites across the United States.

The clinical performance of the Cervista TM HPV 16/18 test was measured against colposcopy and histology results. Biopsy samples were collected from the women with ASO-US cytology as warranted by standard of care guidelines at each participating clinical site. Consensus histology results provided by a central pathologist review panel served as the "gold standard" for determining the presence or absence of disease. In the absence of histology data, the lack of colposcopically visible cervical lesions and no biopsy equated to the absence of disease.

There were 1,312 ASC-UIS subjects with known disease status (central histology or negative colposcopy) and CervistaTm HPV HR and Cervista Tm HPV 16/18 results. The clinical performance characteristics of the Cervista Tm HPV 16/18 test are shown in Tables 9-15.

17 of39 Table 9: Cervista TM HPV 16/18 Results as Compared to Colposcopy/Central Histology Results among Women with ASC-US Cytology

Disease(Central Histology) Cervista TM HPV Cervista TM HPV 16118 Neg HR Result Result Colpascopy No Colposcopy CIN I CIN 2 CIN 3 No Biopsy CIN HPV 16 Positive 39 83 40 25 14 201 HPV HR HPV 18 Positive 11 22 9 0 1 43 Positive HPV 16&18 Both Positive 1 3 5 2 2 13 HPV 16&18 Both Negative 109 273 98 15 5 500 HPV HR HPV 16 and/or 18 Positive 3 3 1 0 0 7 Negative HPV 16&18 Both Negative 210 304 29 5 0 548 Total 373 688 182 47 22 1312

Among those with Cervista MT HPV HR determinate results and disease status data, percent of Indeterminate TM Cervista HPV 16/18 results in the clinical study of women with ASC-US cytology was 0% (0/1312) with 95% Cl: 0% to 0.3%.

Table 10: CervistaTM HPV 16/18 versus Colposcopy/Consensus Histology Results (>CIN2), among Women with ASC-US Cytology

Cervista TM HPV HR Cervista T M HPV 16/18 Result Ž C2 Total Result Positive Negative HPV 16 Positive 39 162 201 HPV 18 Positive 1 42 43 HPV HR Positive -- -~ HPV 16&18 Both Positive 4 9 13 HPV 16&18 Both Negative 20 480 500 HPV 16 and/or 18 Positive 0 7 7 HPV HR Negative HPV 16&18 Both Negative 5 543 548 Total 69 1243 1312

Table 11: Risks of > CIN2 for Different Outcomes of Cervista T M HPV HR and Cervista T M HPV16/18 Tests Prevalence of > CIN2: 5.3% CervistaTM HPV HR Cervista T M HPV 16/18 Likelihood Result Result Risk 95% Cl Ratio 95% CI HPV 16 and/or 18 Positive 17.1% HPV HR (44/257) 13.0% 22.2% 3.72 2.93 4.54 Positive HPVPositiveH 1611861 Negativeeaie4.0% (20/500) 2.6% 6.1% 0.75 0.51 1.06 HPV HR 0.9% Negative HPNega/ive egative (5/555) 0.4% 2,1% 0.17 0.07 0.36

18 of 39 Table 12: Performance of the Cervista TM HPV16/18 Test for Women with CervistaTM HPV HR Positive Results: Prevalence of >CIN2 among Women with CervistaTM HPV Positive Results: 8.5% 95% Cl Sensitivity 68.8% (44/64) 56.6% to 78.8% Specificity 69.3% (480/693) 65.7% to 72.6%

Table 13: CervistaTM HPV 16/18 versus Colposcopy / Consensus Histology Results (> CIN3), among Women with ASC-US Cytology Cervista' m HPV HR CervistaT M HPV 16/18 Result > CIN3 Total Result Positive Negative HPV HR Positive HPV 16 Positive 14 187 201 HPV 18 Positive 1 42 43 HPV 16&18 Both Positive 2 11 13 HPV 16&18 Both Negative 5 495 500 HPV HR Negative HPV 16 and/or 18 Positive 0 7 7 HPV 16&18 Both Negative 0 548 548

Total 22 1290 1312

Table 14: Risks of >CIN3 for Different Outcomes of CervistaTM HPV HR and Cervista TM HPV16/18 Tests Prevalence of >CIN3: 1.7% CervistaTM HPV Cervista TM HPV 16118 Likelihood HR Result Result Risk 95%CI Ratio 95%Ci HPV 16 and/or 18 6.6% HPVHRPos1Positive (17/257) 4.2% 10.3% 4.15 2.99 5.08 HPV 16118 Negative 1.0% (5/500) 0.4% 2.3% 0.59 0.26 1.14 HPV HR 0.0% HPVHR ~HPV 16118 Negative 00 _ _ Negative H0/555 0.0% 0.7% 0.00 0.00 0.37

Table 15: Performance of the CervistaTM HPV16/18 Test for Women with CervistaTM HPV HR Positive Results: Prevalence of >CIN3 among the Subjects with CervistaTM HPV Positive Results: 2.9% 95% Cl Sensitivity 77.3%(17/22) 56.6% to 89.9% Specificity 67.3%(495/735) 63.9% to 70.6%

19 of 39 4 Table 16: Clinical Performance of the Cervista Tm HPV 16/18 Test Stratified by Age for Women with Cervista Tm HPV HR Positive Results

Central Histology?> CIN2 Age: 18 to <21 Positive NeaieTotal HPV HR Positive _HPV ~16 and/~or 18 Positive 7 39 46 HPV 16 & 18 Negaiv 254 56 HPV HR Negative 0 23 23 Total 9 116 12 Disease Prevalence*: 8.8% (9/102) 95% ClI Sensitivity: 77.8% (7/9) 40.0%0/ to 9 7.2 % Specificity: 58.1% (54/93) 47.4% to 68.22% Age: 21 to <30 Positive Negative Total HPV HR Positive HPV 16 and/or 18 Positive 21 117 138 __HPV ~16 & 18 Negative 9 ~ 197 20 HPV HR Negative 0 ~ 13 8 13 Total 30 452 482 Disease Prevalence*: 87% 30/344) 95% Cl Sensitivity: 70.0% (21/30) T06%9/ to 85.3%0 Specificity: 62.7% (197/314) 57. 1%to 681 % Age: 30 to <39 ______Positive Negative Total HPV HR Positive HPV 16 and/or 18 Positive 7 30 37 HPV 16 &l18Negti 3 126 12 HPV HR Negative ______3 125 128 Total 13 281 294 Disease Prevalence*: 6.0n%/ (10/166) 95%/C-l~ Sensitivity: 70.0% (7/10) 34.8% to 93.3% Specificity: 80.8% (126/156) 73.7% to 86.6% Age: 39 or older Positive Negative Total HPV HR Positive HPV 16 and/or 18 Positive 9 27 36 ______PV 16 &l8 Negative 6 103 19 HPV HR Negative 2 264 266 Total 17 394 411 Disease Prevalence*: 13%15145) 95% Cl Sensitivity: 60.0% (9/15) 32.3% to 83.7% Specificity: 79.2% (103/130) 71.2% to 85.8% * Prevalence of Ž CIN2 among women with CervistaTm HPV HR Positive Results

IN WOMEN 30 YEARS AND OLDER WITH NILM CYTOLOGY, PERFORMANCE OF THE Tm CERVISTA HPV 16118 TEST AS A REFLEX HPV TEST TO HELP GUIDE PATIENT MANAGEMENT

A longitudinal 3 year post-approval study has been initiated to support the use of the Cervista T HPV 16/18 test as a reflex test in women 30 years of age and older with normal cytology and positive HPV HR test results. The study design is described below, along with preliminary analytical data obtained from the study population at enrollment. This analytical study was used for evaluation of agreement of the Cervista Tm HPV16/18 test with DNA sequencing as a comparator for HPV detection in the ASO-US and NILM Ž30 populations. Approval for this indication is being given prior to completion of the longitudinal studies in light

20 of 39 C of the analytical study results. Additionally, consistent data obtained from multiple cross- sectional and prospective cohort studies conducted with a variety of cell sampling methods and utilizing a variety of HPV DNA testing methods (both FDA approved, and research grade) provide strong evidence that a negative HPV DNA test implies very low risk of prevalent or incipient CIN 2-3 or cancer when cervical cytology are normal.58'112"23 Furthermore, the absence of HPV 16 and 18 in this population of women further reduces the risk of developing cervical disease and conversely the presence of HPV 16 or HPV 18 augments the relative risk of cervical disease among women >30 years of age regardless of NILM cytological find ings. 10 1, 1 ,13,24 Description of NILM2!3O clinical study Approximately 2,000 qualified subjects with normal Pap test results (NILM) have been enrolled from 26 active clinical centers throughout the United States. It is anticipated that not less than 1,000 subjects will have 3-year follow-up data. The subject retention rate at the end of the first year of follow-up has been nearly 80%. Subjects will be followed for 3 years and have annual study visits. At each follow-up visit, a cervical cytology test is performed. Women who have ASC-US or higher grade cytology results will have a colposcopy performed, and subsequently a biopsy if needed. Analysis of these data will focus on the three-year risk of cervical disease associated with NILM subjects positive for Cervista TM HPV 16/18 as compared to those negative for the test at the time of enrollment (To) and also the three-year risk of cervical disease associated with NILM subjects positive for CervistaTM HPV 16/18 as compared to those negative for any HPV high-risk type at the time of enrollment (To). The presence or absence of HPV at To, will be compared against the presence or absence of (a) > CIN2 and (b) > CIN3 throughout the study. The presence of CIN2, CIN3 or cervical cancer will be ascertained by central histology. Negative results will be defined by colposcopy unless central histology results are available to supersede an initial positive colposcopic indication. All histological interpretation will be conducted by a central pathology review panel.

Comparison of DNA Sequencing and Cervista Tm HPVI 6II8 for the ASC-US and NILM Ž 30 Populations

Residual DNA samples from both the ASO-US and NILM subjects were used for PCR amplification and sequencing. DNA samples were amplified using consensus primers for the HPV Li gene. A portion of the human beta-globin gene was also amplified as an internal control. Purified amplicons were used as templates in multiple sequencing reactions for 14 high-risk types of HPV: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. The sequencing data was analyzed using various sequence alignment software. Below is a comparison between Cervista Tm HPV 16/18 and DNA sequencing for the detection of HPV 16 and l8 in both ASC-US and NILM Ž 30 populations:

Table 17: Performance of CervistaTM HPV16/18 and PCR Sequencing Results, NILM >30

PR Sequencing Mult. HR ce~istaTResultHR HR OeH yeToH ye ye oa CervstamReult Indeterminate Negative - ToH ye ye oa her16&8 ~16& 1&16&

HPV HR Positive

21of 39 HPV 16 Positive 1 48 8 0 6 0 2 0 0 1 66 HPV 18 Positive 0 2 0 6 0 0 0 0 0 0 8 HPV 16&18 Both 0 1 0 0 1 0 0 0 0 0 2 Positive HPV 16118 Negative 12 203 1 0 60 0 0 o 3 0 279 HPV HR Negative HPV 16 Positive 0 12 1 0 0 0 HPV18 Positive 0 6 0 0 0 0 0 0 0 0 6 HPV 16/18 Negative 39 1512 2 0 6 0 0 0 0 0 1559 Total 52 1784 12 673 20 2 0 3 I 1933 Among those with Cervista' m HPV HR determinate results and PCR Sequencing samples, percent of Indeterminate CervistaTM HPV 16/18 results for women with NILM cytology was 0% (0/1933) with 95% Cl: 0% to 0.2%.

Table 18: Comparison of Cervista MT HPV16 and/or HPV18 Results vs PCR Sequencing Results for Women with Cervista TM HPV HR Positive Results, NILM >30

PCR Sequencing HR Positive HPV16 and/or HPV 18 Total Cervista' M HPV HR Positive: Positive Negative Cervista'TM HPV 16/18 Positive 17 58 75

Cervista'T m HPV 16118 Negative 1 266 267

Total 18 324 342

Positive Percent Agreement and Negative Percent Agreement Agreement Percent 95% Ci

Positive % Agreement 94.4% (17/18) 74.2% 99.0%

Negative % Agreement 82.1% (266/324) 77.6% 85.9%

Table 19: Performance ofCervista MT HPV16/18 and PCR Sequencing Results, ASC-US PCR Sequencing One HR Type Two HIR Types Multiple HR Type CervistaTmHRTM HR HR 16 Cervista Result IND Negative & 18

______16 18 Other 16&18 Other Other Others Other Other Other HPV HR Positive HPV16Positive 7 25 96 0 41 0 29 0 1 0 6 0 HPVlePositive 0 0 0 27 7 0 0 9 0 0 0 0 HPV 16&18 Both Positive 0 1 2 0 1 2 1 1 0 3 0 1 HPV 16&18 Both 3 Negative 32 95 6 2 335 0 1 0 27 0 2 0

22 of 39 HPV 16118 Indeterminate 2 1 0 0 7 0 0 HPV HR Negative HPV 16 Positive 1 3 2 0 0 0 0 0 0 0 HPV 16&18 Both Negative 35 510 9 0 10 0 0 0 1 0 0 0

HPV 16118 _ Indeterminate 1 5 0 0 1 0 0 0 0 0 0 0 Total 78 640 115 29 402 2 31 10 31 3 8 1

Among those with Cervista TM HPV HR determinate results and PCR Sequencing samples, percent of Indeterminate Cervista'TM HPV 16/18 results for women with ASC-US cytology was 1.4% (19/1354) with 95% Cl: 0.9% to 2.2%.

Table 20: Comparison of CervistaTM HPV16 and/or HPV18 Results vs PCR Sequencing Results for Women with CervistaTM HPV HR Positive Results, ASC-US

PCR Sequencing, HR Positive HPV16 and/or HPV 18 Total Cervista'T m HPV HR Positive: Positive Negative Cervista'T m HPV 16/18 Positive 177 77 254 Cervista' M HPV 16/18 Negative 11 460 471 Total 188 537 725

Positive Percent Agreement and Negative Percent Agreement Agreement Percent 95% Score Cl Positive % Agreement 94.1% 177/188 89.8% 96.7% Negative % Agreement 85.7% (460/537) 82.4% 88.4%

Analytical Sensitivity Cloned HPV plasmid DNA, representing the HPV types 16 and 18 detected by the CervistaTM HPV 16/18 test, was tested to determine the individual analytical sensitivity for each specific type. Nine HPV-negative characterized DNA samples isolated from cervical specimens were tested in replicates of eight (9 samples x 8 replicates/sample = 72 data points) to determine the Limit of Blank (LoB). The LoB values (FAM FOZ) were 1.18 and 1.21 from HPV 16 and HPV 18 respectively. Limit of Detection (LoD) is the lowest amount of analyte in a sample that the sample has the test results "HPV 16 or HPV 18 detected" at least 95% of the time (results of the test are above the analytical cutoff 95% of the time). Individual Limit of Detection (LoD) values were calculated for both HPV types (16, 18). Each HPV plasmid DNA was tested at concentrations of 5000, 2500, 1250, and 625 copies per reaction, each in a background of three genomic DNA concentrations isolated from an HPV-negative cell line

23 of 39 (10 ng, 100 ng, and 1 /pg per reaction). All positive samples were tested in replicates of eight resulting in 24 replicates per HPV plasmid DNA concentration. The LoB and LoD were evaluated according to the CLSI document EP17-A. 25 The Limit of Detection for each HPV type is referenced in Table 21. Limits are described in terms of the FAM FOZ and as a copy number range. Copy number per reaction LoD values were reported as the copy number range in which 95% of the observed FAM FOZ values were above the LoB.

Table 21: CervistaTM HPV 16/18 Test Analytical Sensitivity Summary LoD LoD HPV DNA Type (Copy Number/Reaction) (FAM FOZ) SDS 16 625-1250 1.34 0.10 18 625-1250 1.33 0.07 In addition to the analytical sensitivity study described above, cell line dilutions were prepared to evaluate the performance of the HPV 16/18 assay using two HPV positive cell lines (HeLa and SiHa) diluted with a HPV negative cell line (Jurkat) to a final concentration of 100,000 cells/ml in PreservCyt media. DNA was isolated from the cell line samples using the Genfind TM DNA Extraction Kit. Using a clinical HPV 16 and HPV18 FOZ cut-off of 2.13, concentrations of approximately 2,500 cells/ml for both SiHa and HeLa cells were above the clinical cutoff 95% of the time.

Clinical Cutoff of the CervistaTM HPV 16118 test The clinical cut-off was evaluated based on HPV16/18 test results targeting a 5% positive rate in the NILM >30 population from a multi-center clinical study. The 95th percentile of the maximum HPV16 and HPV18 FOZ values was determined for NILM >30 subjects and based on this analysis, a FOZ value of >2.13 was selected as the positive cutoff value for the CervistaTM HPV 16/18 test.

Precision Repeatability and within-laboratory precision of the CervistaTM HPV 16/18 test was demonstrated in a 21-day study with three alternating operators, each performing two runs per day on individually assigned sets of equipment. Each run consisted of one plate. Different plate layouts were used for the runs within a day. The procedure followed CLSI EP5-A2. Each run consisted of genomic DNA samples isolated from two HPV positive cell lines (SiHa - Type 16 and HeLa - Type 18), a HPV negative cell line (Jurkat) and contrived samples containing HPV16 or HPV18 plasmid DNA and Jurkat DNA. Each sample was tested in duplicate. The total number of measurements per sample was 84 (21 days, 2 runs per day, 2 replicates per run).

24 of 39 The repeatability and within-laboratory precision values were calculated for each target at each concentration. The precision values for HPV16 FOZ are shown in Table 22 and the HPV18 FOZ values are shown in Table 23. A summary of positive HPV16 and positive HPV18 results are shown in Tables 24 and 25 respectively.

Table 22: HPV 16 Precision Values for Each Target and Concentration Mean Total i-PV Within-Run Between- Between- Between- (Within-lab Copies/Reaction' 16 (repeatability) Run Da 0eatr Precisio~n) Target or CeIlsImnL N FOZ SD %cv SD %CV SD %cv SD %cv S %C HPV16 SOO,000 84 3.708 0.196 5% 0.238 6% 0.348 9% 0.364 10% 0.411 11% ______20,0003 84 7.397 0.697 9% 0.460 6% 0.390 5% 0.331 4% 0.708 10% HPV18 ~~5,000a 84 1.021 0.028 3% 0.042 4% 0.031 3% 0.027 3% 0.047 5% ______20,000a 84 1.024 0.041 4% 0.069 7% 0.045 4% 0.048 5% 0.073 7% 5000 SiHa I 95,000 SiHa/Jurkat - Jurkat' 84 2.430 0.160 7% 0.115 5% 0.138 6% 0.135 6% 0.196 8% 20,000 SiHa I ______80,000 Jurkat b 84 5.465 0.220 4% 0.360 7% 0.384 7% 0.324 6% 0.486 9% 2500 HeLa / Hela/Jurkat 97,500 Jurkatb 84 0.784 0.029 4% 0.047 6% 0.049 6% 0.048 6% 0.063 8% 10,000 HeLa / 90,000 Jurkatb 84 0.893 0.037 4% 0.037 4% 0.039 4% 0.036 4% 0.053 6% ______b1 _84 10.886 0.111 12% 0.074 8%6 0.064 7% 0.029 3% 0.114 13% Jurkat 80004 0.870 0.029 3% 0.03 4 030 3% 0.023 3% 0.044 5%

______100,000b 84 .0 l170.066. 7% 0.04 % 04 5% 0.039 4% 07 8% aHPV1G or HPV18 plasmid DNA at the indicated concentration (copies/reaction) mixed with lO0ng/reaction of HPV negative genomic DNA (Jurkat).

bGenomnic DNA isolated from HPV positive cells (SiHa and HeLa) and/or HPV negative cells (Jurkat) at the indicated concentration (cells/mL).

Table 23: HPV 18 Precision Values for Each Target and Concentration Mean Within-Run Between- Betwen- Between- Total (Within- HPV (repeatability) Run Day Oprtor lab precision) Copies/Reaction 18 ___ Target or Cells/mL b N FOZ SD %Cv SD %CV SD %CV SD %cv SD %Cv HPV 16 5,OO0a 84 0.978 0.041 4% 0.055 6% 0.059 6% 0.050 5% 0.076 8% 20,000a 84 0.990 0.055 6% 0.068 7% 0.062 6% 0.043 4% 0.087 9% HPV 18 5CO0s 84 3.620 0.243 7% 0.255 7% 0.265 7% 0.230 6% 0.363 10% ______20,000a 84 8.483 0.396 5% 0.613 7% 0.595 7% 0.378 4% 0.787 9% 5000 SiHa / 95,000 SiHa/Jurkat Jurkat 84 0.874 0.051 6% 0.035 4% 0.045 5% 0.043 5% 0.062 7% 20,000 SiHa/ ______80,000 Jurkat b 84 0.858 0.023 3% 0.052 6% 0.043 5% 0.044 5% 0.059 7% 2500 HeLa / 97,500 Hela/Jurkat Jurkath 84 2.988 0.163 5% 0.174 6% 0.175 6% 0.064 2% 0.243 8% 10,000 HeLa I 90,O000Jurkat______84 7.1 .2 % 146 19% 1.757 22% 0.463 6%/ 2.062 26% 10,000 ~84 0.927 0.055 6%9 0.05 6 % 0.0.55 6% 0.043 5% 0.077 8% Jurkat b 2 0 0 8 090 003 4% 008 4 0035 4% 0.027 3% 0.051 6%

______~49%/ 10008 .5 0031.5 %002 4 .363% 0.060 6%

25of 39 a HPV16 or HPV18 plasmid DNA at the indicated concentration (copies/reaction) mixed with 1OOng/reaction of HPV negative genomic DNA (Jurkat). bGenomic DNA isolated from HPV positive cells (SiHa and HeLa) and/or HPV negative cells (Jurkat) at the indicated concentration (cells/mL).

Table 24. Summary of Positive HPV16 Results for Precision Study. Copies/Reactiona Mean HPV 16 Positive % (n) Target or N HPV 16 Operator Operator Operator Cells/mLb FOZ 1 2 3 Total .5,0008 84 3.708 100% 100% 100% 100% HPV 16 ___3_70 (28)) (84 20,000a 84 7.397 100% 100% 100% 100% (28) (28) ' (28) (84) a 5,000 84 1.021 0% (0) 0% (0) 0% (0) 0% (0) HPV 18 a I 20,000 84 1.024 0% (0) 0% (0) 0% (0) 0% (0) 5000 SiHa / 95,000 (23) Jurkatb 84 2.430 82% (3 100% 100% 94% SiHa/Jurkat Si~a/urkatJurkat b(28) (28) (79) 20,000 SiHa / 80,000 100% 100% 100% 100% Jurkatb 84 5.465 (28) (28) (28) (84)

2500 HeLa / b97,'500 8 Jurkat 84 0.784 0% (0) 0% (0) 0% (0) 0% (0) Hela/Jurkat 10,000 HeLa / 90,000 84 0.893 0% (0) 0% (0) 0% (0) 0% ______Jurkatb (0) b 10,000 ___ _ 84 0.886 0%(0) 0% (0) 0% (0) 0% (0) Jurkat 20,000 b 84 0.870 0% (0) 0% (0) 0% (0) 0% (0) b 100,000 1 84 0.917 0% (0) 0% (0) 0% (0) 0% (0) HPV16 or HPV18 plasmid DNA at the indicated concentration (copies/reaction) mixed with 10ing/reaction of HPV negative genomic DNA (Jurkat). bGenomic DNA isolated from HPV positive cells (SiHa and HeLa) and/or HPV negative cells (Jurkat) at the indicated concentration (cells/mL).

Table 25. Summary of Positive HPV18 Results for Precision Study. Copis/ReorMean oa HPV Target Copies/Reaction a or 18 Positive % (n) Target Cells/mLb N HPV 18 Operator Operator Operator FOZ 1 2 3 Total a 5,000 84 0.978 0% (0) 0% (0) 0% (0) 0% (0) HPV 16 20,000a 84 0.990 0% (0) 0% (0) 0% (0) 0% (0) 100% 100% 100% 100% HPV 18 5 328(84) (28) (28) 20,000a 84 8.483 100% 100% 100% 100% (28) (28) (28) (84) 5000 SiHa / 95,000 84 0.874 0% (0) 0% (0) 0% (0) 0% (0) SiHa/Jurkat Jurkat 20,000 SiHa / 80,000 84 0.858 0% (0) 0% (0) 0% (0) 0% (0) Jurkat b Hela/Jurkat 2500 HeLa / 97,500 84 2.988 100% 100% 100% 100% Jurkatb______(28) (28) (28) (84)

26 of 39 (s7 10,000 HeLa / 90,00 100% 100% 95% ______~Jurkatb 84 7.918 (28) (28) 86% (24) (80) 10,000b84 0.927 0%(0) 0%(0) 0%(0) 0%(0)

Jurkat 20 0b84 0.920 0% (0) 0% (0) 0% (0) 0% (0) I 100.0001 ~ 84 10.951 0% (0) I0% (0) I0% (0) I0% (0)

8HPV16 or HPV18 plasmid DNA at the indicated concentration (copies/reaction) mixed with lO0ng/reaction of HPV negative genomic DNA (Jurkat). bGenomic DNA isolated from HPV positive cells (SiHa and HeLa) and/or HPV negative cells (Jurkat) at the indicated concentration (cells/mL).

Reproducibility Reproducibility of the CervistaiMr HPV 16/18 test was assessed at three external sites using a panel of HPV positive and negative cultured cells and HPV positive and negative cervical specimens. DNA was extracted from 2 mL of cervical specimen or cultured cells suspended in PreservCyt® Solution. The DNA was extracted using the Genfind Tm DNA Extraction Kit. Sixteen samples were extracted for DNA and tested with Cervista Tm HPV 16/18 at three locations on five non-consecutive days within a two-week time period. Two lots of Cervista Tm HPV 16/18 kits and three lots of Genfind Tm DNA Extraction Kits were used across the 3 sites for the study. The total number of measurements for each sample was 15 = (3 sites x 5 days x 1 run per day). A summary of the percent agreement between the expected and observed results combined for all sites is shown in Table 26. A summary of individual sample results across sites with a cumulative mean, standard deviation and 95% confidence interval for the HPV16 and HPV18 FOZ values are presented in Table 27 and Table 28. Table 26. Data Summary for a Multi-center Reproducibility Study of the CervistaTm HPV

16/18 Test. ______Expected Number of Results in PretAemnt Lower Limit of Result Results Agreement PretAemnt95% cl

Positive 150 150 100.0% 97.5%

Negative 90 90 100.0% 95.9%

Table 27. Summary of Cervista Tm HPV16 Results from a Multi-Center Reproducibility Study Sample Type HPV 16 FOZ HPV 16 Positv n ___ and

Sample Cocnrtn N Mean SD Site I Site 2 Site 3 Tota

1Neg 100,000 Jurkat 15 0.899 0.048 0(0) 0 (0) 0 (0) 0 2Pos:HPV18 10,000 HeLa 2 Pos:HPV18__ 90,0600 Jurkat 15 0.883 0.076 0 (0) 0 (0) 0 (0) 0 5,000 He~ ___ 3 Pos:HPV18 95000Jukt 1 0.847 0.083 0(0) 0 (0) 0 (0) I 0

27 of 39 2,500 HeLa 4 Pos:HPV18 97500 JLa 15 0.833 0.073 97,500 Jurkat 0 (0) 0 (0) 0 (0) 0 20,000 SiHa 80,000 Jurkat 15 6.345 0.553 100 (5) 100 (5) 100 (5) 15 10,000 SiHa 6 Pos:HPV16 90,000 Jurkat 15 4.933 0.598 100 (5) 100 (5) 100 (5) 15 5,000 SiHa 7 Pos:HPV16 95,000 Jurkat 15 3.049 0.473 100 (5) 100 (5) 100 (5) 15 5,000 SiHa S Pos:HPV18 2,500 HeLa 15 3.047 0.387 100 (5) 100 (5) 100 (5) 15 and HPV16 12,500 Jurkat 9 Neg Cervical Pool 15 0.905 0.078 0 (0) (0)0 (0)0 0 10 Neg Cervical Pool 15 0.888 0.097 0 (0) 0 0 0(0) 0 11 Pos:HPV18 Cervical Pool 15 0.958 15 0.9580.154 0.154 0 (0) 0 (0)0___ (0o )0_ 12 Neg Cervical Pool 15 0.865 0.127 0 13 HPV16 Cervical Pool 15 9.769 0.658 100 (5)100 5 100(5 15 14 Neg Cervical Pool 15 0.919 0.093 0 0 0 0 0 0 15 HPV16 Cervical Pool 15 2.782 0.611 100 5 100 5) 100 5 15 16 Neg Cervical Pool 15 1.049 0.130 0 (0) 0 (0) 0 (0) 0

Table 28. Summary of CervistaTM HPV18 Results from a Multi-Center Reproducibility Study Sample Type and HPV 18 FOZ HPV18 Positive % n) Concentration Sample N Mean SD Site 1 Site 2 Site 3 Total ______(cells/m i) 1 Neg 100,000 Jurkat 15 0.927 0.042 0 (0) 0 0 10,000 HeLa 2Pos:HPV18 90,000 Jura 15 9.322 0.831 100 (5) 90,0600 Jurkat 10 100 (5) 15 5,000 HeLa 3 Pos:HPV18 95,000 Jurkat 15 6.121 1.105 100 (5) 100 (5) 100 (5) 15 2,500 HeLa 4Pos:HPV18 97500 Jura 15 3.645 0.455 100 (5) 100 (5) 100 (5) 15 20,500 Jurkat

10,000 SiHa 6 Pos:HPV16 90,000 Jurkat 15 0.963 0.043 0(0) 0 (0) 0(0) 0 50,000 SiHa 7 Pos:HPV16 95,000 Jurkat 15 0.927 0.031 0 (0) 0 (0) 0 (0) 0 5,000 SiHa 8 Pos:HPV18 2,500 HeLa 15 3.815 0.435 100 (5) 100 (5) 100 (5) and HPV16 12,500 Jurkat 15

9 Neg Cervical Pool 15 0.896 0.049 0 (0) 0 (0) 0 (0) 0 10 Neg Cervical Pool 15 0.892 0.053 0 0(0(0) 0 0 11 Pos:HPV18 Cervical Pool 15 10.413 1.945 100(5 100 5 100 (5) 15 12 Neg Cervical Pool 15 1.146 0.121 9j0 0(0) 0 0 0

28 of 39 13 HPV16 Cervical Pool 0.861 0.053 00 0 14 Neg Cervical Pool 0.927 0.029 00000 0 15 HPV16 Cervical Pool 15 0.921 0.035 0 0 0 0 0(00 16 Neg Cervical Pool 15 0.921 0.050 0 (0) 0

Interfering Substances Three cell-line samples (one HPV negative, one HPV16 positive, one HPV18 positive) described in Table 29 were tested with interferents that could potentially be present in the cervical specimen or transferred inadvertently during sample extraction using the GenfindTM DNA Extraction Kit (Table 30). Concentration levels were chosen to represent extreme conditions that could potentially occur during specimen collection if the cervix was not cleared prior to obtaining the specimen. DNA was isolated from pure and impure samples using the GenfindTM DNA Extraction Kit and was tested with the CervistaTM HPV 16/18 test to assess interference caused by the introduced substances.

Table 29: Interfering Substances Sample Descriptions Sample Description Cell line sample stored in PreservCyt solution containing 1001000 cells/mL Jurkat (HPV J Negative) cells Cell line sample stored in PreservCyt solution containing 7,500 cells/mL SiHa cells (HPV 16 SiHa/Jurkat Positive) and 92,500 cells/mL Jurkat cells Cell line sample stored in PreservCyt solution containing 2,500 cells/mL HeLa cells (HPV 18 HeLa/Jurkat Positive) and 97,500 cells/mL Jurkat cells

Table 30: Interference Results Interferent Concentrations Interference Source Type Tested Observed? Blood Visually Detectable No Mucous Visually Detectable No Blood/Mucous Visually Detectable No Vaginal Douche 0.5%, 2% No Cervical Specimen ContraceptiveJelly 0.5%,2% Yes" Anti-fungal Cream containing2% clotrimizole 0.5%, 2% Ys Anti-fungal Cream containing 4% miconazole 05%, 2% PreservCyt'' Solution 0.5%, 2% GenfindTM DNA Extraction Kit 7E n%0 No Sample Processing Mgt______5%,_1 Ead51 0 % No I Magnetic Beads 1 5%, 10% I No 'The levels of interferent required to cause testing failures (2%) are unusually high and should not be encountered in actual clinical specimens. During DNA extraction, the contraceptive jelly showed visually detectable interferance with the magnetic bead separation in the 10 mM Tris buffer, causing low DNA recovery and insufficient DNA sample for testing.

29 of 39 7t) The levels of interferent required to cause testing failures are unusually high and should not be encountered in actual clinical specimens if the clinician follows the proper cervical cytology sampling procedure of clearing the cervix before obtaining the cell sample for cervical cytology.

Cross-Reactivity A panel of bacteria, fungi, and viruses commonly found in the female anogenital tract, as well as several Human papillomavirus types of high, low, or undetermined risk were tested with the CervistaTM HPV 16/18 test to assess potential cross-reactivity. Table 31: The organisms listed below were added to PreservCyt® Solution at concentrations of approximately 1 x10 5 cfu/mL and 1x10 7 cfu/mL. DNA from these organisms and a negative cell line (Jurkat, 1x10 5 cells/mL) was extracted using the GenfindTM DNA Extraction Kit. All samples yielded negative results with the CervistaTM HPV 16/18 test. Candida albicans Proteus vulgar/s Co nebacterium seudodiptherit/cum Staphylococcus aureus Enterococcus faecalis Staphylococcus epideridis Escherichia co/i Streptococcus mit/s Lactobacillusac/dophi/us yoenesStreptococcus

Table 32: Purified DNA obtained from the or anisms listed below was tested at 5 concentrations of 1x10 copies/reaction and 1x10 copies/reaction using the CervistaTM HPV 16/18 test. All samples yielded negative results. Herpes simplex virus, type 1 (HSV-1) Chlamydia trachomatis Herpes simplex virus, type 2 (HSV-2) Neisseria gonorrhoeae Human Immunodeficiency Virus type 1 Neisseria meningitides (HIV-1, pot and env regions) Mycoplasma hominis

Table 33: Cloned DNA or PCR amplicons for the following samples were tested at 5 concentrations of 1x10 copies/reaction and 1x107 copies/reaction unless noted, using the Cervista TM HPV 16/18 test. All samples yielded negative results. Human papillomavirus type la Human apillomavirus type 51 Human papillomavirus type 6 Human papillomavirus type 52 Human papillomavirus type 11 Human papillomavirus type 53 Human papillomavirus type 31a Human papillomavirus type 58 Human pap uman papillomavirus type 59 Human papillomavirus type 39 Human papillomavirus type 66 Human papillomavirus type 42 Human papillomavirus type 67 Human apillomavirus tpe 43 Human Dapillomavirus type 68 Human papillomavirus type 44 Human papillomavirus type 70 Human papillomavirus type45 Human Internal Control gene aHuman papillomavirus type 31 yielded TM 7 positive HPV16 results with the Cervista HPV 16/18 test at 1X10 copies/reaction. Upon further titration T of the HPV 31 sample, negative results were obtained with the CervistaM HPV 16/18 test at 1x106 copies/reaction,

An additional cross-reactivity study was conducted for Chlamydia trachomatis,, Neisseria gonorrhoeae, Neisseria meningitides, and Mycoplasma hominis utilizing

30 of 39 whole organisms spiked into PreservCyt® Solution containing HPV-negative Jurkat Cells (100,000 cells/ml). Three lots of each organism were prepared and DNA was isolated from all samples using the Genfind TM DNA Extraction kit. This study demonstrated that the Cervista TM HPV 16/18 test does not cross-react with DNA isolated from PreservCyt® samples containing up to containing up to 1.Ox1 0 7 cfu/ml of Neisseria meningitides and Mycoplasma hominis, 5x1 06 cfu/ml of Neisseria gonorrhoeae and 1.Ox1 06 cfu/ml Chlamydia trachomatis.

REFERENCES 1. American Society for Colposcopy and Cervical Pathology website: www.asccp.org, 2008. 2. National Cancer Institute website: www.cancer.qov, 2008. 3. Meijer CJ, Snijders PJ, and Castle PE. 2006. Clinical utility of HPV genotyping. Gynecol Oncol 103: 12-17. 4. Winer RL, N Kiviat, J Hughes, D Adams, S Lee, J Kuypers, L Koutsky. 2005. Development and duration of human papillomavirus lesions, after initial infection. J Infect Dis 191: 731-738. 5. Schlecht NF, Kulaga S, Robitaille J, et al. 2001. Persistant human papillomavirus infection as a predictor of cervical intraepithelial neoplasia. JAMA 286: 3106-3114. 6. Wright TC, Jr., Cox JT, Massad LS, Twiggs LB, Wilkinson EJ. 2002. 2001 consensus guidelines for the management of women with cervical cytological abnormalities. JAMA 287: 2120-2129. 7. Solomon D, Schiffman M, and Tarone R. 2001. Comparison of three management strategies for patients with atypical squamous cells of undetermined significance: baseline results from a randomized trial. Jour Nat Can Inst 93(4): 293-299. 8. Sherman ME, Schiffman M, and Cox TJ. 2002. Effects of age and human papilloma viral load on colposcopy triage: data from the randomized Atypical Squamous Cells of Undetermined Significance/Low-grade Squamous Intraepithelial Lesion Triage Study (ALTS). Jour Nat Can Inst 94(2): 102-107. 9. Davey DD, Neal MH, Wilbur DC, Colgan TJ, Styer PE, and Mody DR. 2004. Bethesda 2001 implementation and reporting rates: 2003 practices of participants in the college of American Pathologists Interlaboratory Comparison Program in Cervicovaginal Cytology. Arch Path Lab Med 128: 1224-1229. 10. Khan MJ, Castle PE, Lorincz AT, et al. 2005. The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst 97:1072-1079. 11. Castle PE et al 2005. Human Papillomavirus Type 16 Infections and 2-Year Absolute Risk of Cervical Precancer in Women With Equivocal or Mild Cytologic Abonormalities. J Natl Cancer Inst 97: 1066-1071. 12. Mayrand MH, E Duarte-Franco, I Rodrigues, SD Walter, J Hanley. 2007. A Ferenczy, S Ratnam, F Coutlee, EL Franco. Human papillomavirus DNA versus Papanicolau screening tests for cervical cancer. N Engl J Med 357(16): 1579-1588. 13. Wheeler CM, WC Hunt, M Schiffman, PE Castle. 2006. Human papillomavirus genotypes and the cumulative 2-Year risk of cervical cancer. J Infect Dis 194: 1291- 1299.

31 of39 14. Bosch X and Harper D. 2006. Prevention strategies of cervical cancer in the HPV vaccine era. Gynecol Oncol 103(1): 21-24. 15. Bulkmans NW, Berkhof J, Bulk S, Bleeker MC, van Kemenade FJ, Rozendaal L, Snijders PJ, Meijer CJ; POBASCAM Study Group. 2007. High-risk HPV type-specific clearance rates in cervical screening. Br J Cancer 96(9): 1419-1424. 16. Sandadi S, RL Flyckt, KM Zanotti. 2008. Cost effectiveness of a triage strategy for atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion cytology using human papillomavirus 16/18 genotyping. Gynecological Oncol 108(S96): 218. 17. Saslow D, Runowicz CD, Solomon D, Moscicki A-B, Smith RA, Eyre HJ, Cohen C. 2002. American Cancer Society guideline for the early detection of cervical neoplasia and cancer. CA Can Jour Clin 53: 342-362. 18. Wright TC, Jr., Schiffman M, Solomon D, Cox JT, Garcia F, Goldie S, et al. 2004. Interim guidance for the use of human papillomavirus DNA testing as an adjunct to cervical cytology for screening. Obstet Gynecol 103: 304-309. 19. Wright TC, Jr., Massad LS, Dunton CJ, Spitzer M, Wilkinson EJ, and Solomon D. 2007. 2006 consensus guidelines for the management of women with abnormal cervical cancer screening tests. Am J Obstet Gynecol 197(4): 346-355. 20. Castellsague X et al. 2006. Worldwide Human Papillomavirus Etiology of Cervical Adenocarcinoma and Its Cofactors: Implications for Screening and Prevention. J Natl Cancer Inst 98: 303-315. 21. Hall JG, Eis PS, Law SM, Reynaldo LP, Prudent JR, Marshall DJ, Allawi HT, Mast AL, Dahlberg JE, Kwiatkowski RW, de Arruda M, Neri BP, and Lyamichev VI. 2000. Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction. PNAS 97(15): 8272-8277. 22. Revzina N, DiClemente R. 2005. Prevalence and incidence of human papillomavirus infection in women in the USA: a systematic review. Int J of STD & AIDS 528-537. 23. Woodman CB, Collins S, Winter H, Bailey A, Ellis J, Prior P, Yates M, Rollason TP, Young LS. 2001. Natural history of cervical human papillomavirus infection in young women: a longitudinal cohort study. Lancet 357(9271): 1831-1836. 24. Woodman CB, Collins S, Rollason TP, Winter H, Bailey A, Yates M, Young LS. 2003. Human papillomavirus type 18 and rapidly progressing cervical intraepithelial neoplasia. 361(9351): 40-43. 25. CLSI document EP17-A: Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline

TABLE 34: TROUBLESHOOTING GUIDE Observation Probable Cause Solutions Insufficient volume made for Number of samples Manually recalculate the required amount of reaction mixes entered in "Assay reaction mix needed to complete the entire Selection" tab of software plate.

32 of 39 .3 Observation Probable CauseSouin is less than samples Recreate software printouts using correct added to the plate. number of samples. Excessreactin mixVerify the correct reaction mix volumes were volume added to 96 well adetoacwl. microplate. Verify the calibration information on equipment is current. _Fluo~rescence mcolt No Target Control displays the reader gain settings are Increase the fluorometer gain settings for the following results: too low causing the raw designated scan(s) so that the No Target * Increase gain for scan 1 fluorescent signal values Control produces a minimum signal of 600 * Increase gain for scan 2 to fall below the minimum RFU and re-read the plate. * Increase gain for both scans requirement. Errors occur during data import: "Check FAM & Red gain settingsSeTrulsotnGidinheIverCl and read the whole plate again. RepeTroublshoftware Gusd Manalfonthnaer (atalplated) rasrentFluorometer issues fluorometer issues that may contribute to this "Check FAM gain setting and err read the whole plate again. (Partial plate reads are not allowed.)" Thermal incubation period Cofrththeicbinwapromdfr "Check Red gain setting and was longer than theCofrththeicbinwapromdfr read the whole plate again, specified length of time the specified length of time and at the (Partial plate reads are not recommended specified temperature. allowed.)"

33 of 39 ij * Be sure all samples, reagents and reaction mixes are mixed thoroughly.

Insufficient or inconsistent * When adding reaction mix to each well, mixing of reagents place tips at the bottom of the well (beneath mineral oil) and slowly pipette up and down 3-4 times.

______Verify all liquid is expelled from the pipette tip during additions. a Verify the correct reagent was added to Incorrect preparation of each well. reaction mixes * Verify the correct reagent volumes were added to each well. * Verify the calibration information on Inconsistent addition of the equipment is current. No Target Control or * Visually inspect plate for consistent volumes reaction mix to the between wells. No Target Control displays the microplate following results: High %Difference (gDNA Use nuclease-free aerosol barrier tips and NTC) sterile tubes when making the reaction mixes,

Wear gloves when setting up the test.

Suspected contamination Since nucleases may be present, make sure during sample addition or that pipette tips do not touch any other reaction mix preparation surfaces except the solution being dispensed.

Do not touch pipette tips with hands.

Clean lab surfaces using appropriate materials. Sample evaporation Verify mineral oil addition to each well. Bubbles in the reaction If possible, spin down plates prior to wells fluorescence scanning. Prepared reaction mixes were not used within Use reaction mixes within 30 minutes of recommended time period preparation.

34 of 39 • Insufficient or inconsistent * Be sure all controls and reagents are mixed mixing of controls toroughly and consistently. * When adding reaction mix to each well, place tips at the bottom of the well (beneath mineral oil) and slowly pipette up and down 3-4 times. * Make sure that all liquid is expelled from the Inconsistent addition of pipette tip during additions. reaction mix * Verify that the correct control was added to each well. * Verify that the correct control volume was added to each well. * Verify the calibration information on equipment. Insufficient or Inconsistent * Visually inspect plate for consistent volumes addition of control between wells.

Correct control(s) was not added to the plate or was Verify the correct controls were added to the Control(s) displays not added to the correct correct plate positions. "Invalid Control" result plate position Incubation period-was shorter or longer than the Confirm that the incubation was performed for specified length of time the specified length of time and at the specified recommended temperature.

Suspected contamination Make sure that pipette tips touch only the during sample addition solution being dispensed.

Do not touch pipette tips with hands.

Clean lab surfaces using appropriate materials. Sample evaporation Verify mineraloil addition toeach well. The plate may not be When scanning the plate, orient the plate so orientated properly well A-i isin the upper left-handcorner. Bubbles in the reaction if possible, spin down plates prior to wells fluorescence scanning. Prepared reaction mixes s ecinmxswti 0mntso were not used withinUsrecinmxswtn30iueso recommended time period preparation.

35of 39 Be sureall samples and reagents are mixed thoroughly. W henadding reactionmix to eachwell, place tips at the bottom of the well (beneath the mineral oil) and slowly pipette up and down 3- 4 times. Insufficient or inconsistent Verify all liquid is expelled from the pipette tip mixing of samples during additions.

Inconsistent addition of Verify the correct sample was added to each reaction mixwel Inconsistent addition of sample Verify the correct sample volume was added to each well.

Verify the calibration information on equipment is current.

Sample displays Visually inspect plate for consistent volumes "IND: High %Difference" result between wells.

Use nuclease-free aerosol barrier tips and sterile tubes during set up.

Wear gloves when setting up the test. Suspected contamination SuringspectedmcontaminatMake sure that pipette tips touch only the duigsiionsolution being dispensed.

Do not touch pipette tips with hands. Clean lab surfaces using appropriate materials. Sample evaporation Verify mineral oil addition to each well. Bubbles in the reaction If possible, spin down plates prior to wells fluorescence scanning. Prepared reaction mixes werePerepartused not usedreacthin withinmi Use reaction mixes within 30 minutes of recommended time period preparation.

36 of 39 Insufficient number of cells a Mix the specimen well and repeat DNA in specimen extraction from the specimen.

Suspected error during * Verify the correct sample volume was added DNA extraction to each well.

Insufficient amount of DNA · Verify that proper procedure was followed was used in the test for DNA extraction. Sample displays "IND: Low gDNA" result Repeat DNA extraction from the specimen. DNA sample inhibition Refer to the Package Insert, Performance Characteristics (Interfering Substances) section. The DNA sample(s) may Verify that the sample was denatured at the not have been completely correct temperature and for an appropriate denatured amount of time. * Repeat DNA extraction from the specimen. Suspected error during DNA extraction a Verify that proper procedure was followed Sample displays for DNA extraction. "IND: Low HPV FOZ" result * Refer to the Package Insert, Performance DNA sample inhibition Characteristics (Interfering Substances) section. * Repeat DNA extraction from the specimen. Insufficient elution volume during DNA extraction · Verify that proper procedure was followed for DNA extraction. Suspected error during High number of DNA samples DNA extraction · Repeat DNA extraction from the specimen. with positive FAM FOZ values in both reaction mixes Suspected DNA extraction * Verify that proper procedure was followed reagent contamination for DNA extraction.

CONTACT INFORMATION

Manufacturer: Third Wave Technologies, Inc., 502 S. Rosa Road, Madison, WI, 53719 USA Phone: 608.273.8933 Website: www.twt.com

Technical Support: Third Wave Technologies, Inc., Phone: 888.898.2357, option 3

37 of 39 NOTICE TO RECIPIENT ABOUT LIMITED LICENSE The receipt of this product from Third Wave Technologies, Inc. or its authorized distributor includes a limited, non-exclusive license under patent rights held by Third Wave Technologies, Inc. Acquisition of this product constitutes acceptance by the recipient of this limited license. Recipients unwilling to accept the limited license must return the product for a full refund. Such license is solely for the purposes of using this product to detect a specific analyte. For avoidance of doubt, the foregoing license does not include rights to use this product for agriculture or veterinary medicine applications. The foregoing license does not include a license to use the product for new product research or development, product manufacture, or any reverse-engineering purposes. The purchaser of this product is not authorized to transfer this product to any third party for any purpose without the express written consent of Third Wave Technologies, Inc. Except as expressly provided in this paragraph, no other license is granted expressly, impliedly, or by estoppel. For information concerning the availability of additional licenses to practice the patented methodologies, contact: Legal Department, Third Wave Technologies, Inc., 502 South Rosa Rd., Madison, WI, 53719, (608) 273-8933.

The Cervista Tm HPV 16/18 test uses a proprietary Invader® chemistry and specific components covered under: U.S. Patent Nos. 5,614,402; 5,795,763; 5,846,717; 5,985,557; 5,994,069; 6,001,567; 6,090,543; 6,090,606; 6,348,314, 6,458,535; 6,555,357; 6,562,611; 6,635,463; 6,673,616; 6,759,226; 6,872,816; 6,875,572; 6,913,881; 7,060,436; 7,067,643; 7,087,381; Canadian Patent Nos. 2,163,015; 2,203,627; Australian Patent Nos. 694,736; 731,062; 737,449; 738,849; 744,369; 779,443; 781,188; Japanese Patent No. 3,665,648; European Patent No. 711,361. All U.S. patents and foreign patents where applicable that have or may hereafter issue in respect of such applications for patents; and all U.S and foreign patent applications and patents issuing thereon where applicable whose subject matter in whole or in part is entitled to the benefit of the filing date(s) of any of the foregoing patents/patent applications listed in this product insert.

LIMITED PRODUCT WARRANTY Third Wave Technologies, Inc. warrants that this product will meet the specifications stated on the product information sheet. If any component of this product does not conform to these specifications, Third Wave Technologies, Inc. will at its sole discretion, as its sole and exclusive liability and as the users sole and exclusive remedy, replace the product at no charge or refund the cost of the product; provided that notice of nonconformance is given to Third Wave Technologies, Inc. within sixty (60) days of receipt of the product.

This warranty limits Third Wave Technologies, Inc. liability to the replacement of this product or refund of the cost of the product. NO OTHER WARRANTIES OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING WITHOUT LIMITATION IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE OR NON- INFRINGMENT, ARE PROVIDED BY THIRD WAVE TECHNOLOGIES, INC. Third Wave Technologies, Inc. shall have no liability for any direct, indirect, consequential or incidental damages arising out of the use, the results of use or the inability to use this product and its components.

38 of 39 7 c1 Third Wave®, Cleavase®, and Invader® are registered trademarks of Third Wave Technologies Inc. CervistaTM and Invader Call Reporter Tm are trademarks of Third Wave Technologies Inc. All other Trademarks / Registered Trademarks referenced within this product insert, are the property of each of their respective companies.

Some components of nucleic acid analysis, such as specific methods and compositions for manipulating or visualizing nucleic acids for analysis, may be covered by one or more patents owned by other parties. Similarly, nucleic acids containing specific nucleotide sequences may be patented. Making, using or selling such components or nucleic acids may require one or more licenses. Nothing in this document should be construed as an authorization or implicit license to make, use or sell any so covered component or nucleic acid under any such patents.

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